Vaccinia Capping System (C09-1082-500)

Product DescriptionThe vaccinia virus capping system uses the vaccinia virus capping enzyme and related components to add the 7-methylguanosine cap structure (m7Gppp, Cap0) to the 5´ end of the RNA. In eukaryotes, this structure is closely related to the stability, transport and translation of mRNA. Using an enzymatic reaction to cap RNA is a simple and effective method, and the cap structure is exactly the same as the natural Cap 0 structure, which can significantly improve the stability and translation ability of RNA used for in vitro transcription, transfection, and microinjection. This enzyme is composed of two subunits (D1 and D12). The D1 subunit performs the functions of RNA triphosphatase and guanosine transferase, and the D12 subunit performs the function of guanine methyltransferase. They are essential for adding a complete The Cap 0 structure m7Gppp5´N is required.Figure 1. The structure and mechanism of vaccinia virus capping. a. Co-crystal structure of vaccinia virus capping enzyme, GTP (red) and SAH (rose red) (PDB 4CKB). The enzyme is composed of two subunits, D1 and D12, and has the functions of RNA triphosphatase (blue), guanosine transferase (orange) and guanine methyltransferase (beige). This figure is quoted from Ramanathan, A., Robb, GB, & Chan, SH (2016). mRNA capping: biological functions and applications. Nucleic Acids Res 44(16), 7511–7526. b. The cap structure of mRNA consists of a 7-Methylguanosine is composed of a 5'-5' triphosphate bridge connected to the 5'nucleoside of the mRNA chain. The Cap-0 structure is formed by the sequential reaction of three adjacent RNA strands. The further formation of cap-1 structure requires the participation of 2'-O-methyltransferase, which can reduce the cellular innate immune response caused by RNA in vivo. This figure is quoted from Decroly, E., Ferron, F., Lescar, J. et al. (2012). Conventional and unconventional mechanisms for capping viral mRNA. Nat Rev Microbiol 10, 51–65.This system can be used for the capping reaction of IVT RNA, so that the capping reaction is completed within 1 hour, the efficiency is close to 100%, and the correct direction is guaranteed.Product UsageIn vivo or in vitro pre-translational capping of mRNA and 5'end labeling of mRNA.PrecautionsSAM: It is necessary to calculate the amount of SAM in advance, and dilute the aliquoted 32 mM stock solution to 2 mM working solution before the reaction starts. SAM is unstable at pH 7-8 and 37°C and needs to be freshly prepared before the reaction starts. To avoid degradation of SAM, this working solution needs to be stored on ice.RNA: Before using the capping system, the RNA produced by the in vitro transcription reaction should be purified and dissolved in RNase-free water. The RNA should not be dissolved in EDTA solution or other salt solutions.RNA secondary structure: Some RNA transcripts may form stable secondary structures (homodimers and hairpin structures). For example, the secondary structure occurring at the 5'end of the transcript will affect the capping efficiency. Heating RNA before reacting with the capping enzyme can remove the secondary structure at the 5´ end of the transcription product. If the 5´ end of the transcription product has a complex structure, the heating time can be extended to 10 minutes, and the capping reaction time can be extended to 60 minutes.Cap 0- and Cap 1-mRNA: The difference between Cap 0- and Cap 1-mRNA is whether the first nucleotide (N1) next to the cap structure at the 5'end of RNA has a 2'-O-methyl group. In higher eukaryotic cells, this methylation is part of the natural capping process, and the Cap 1 structure improves the translation efficiency of mRNA.Poly(A) tail: If the capped RNA needs to be added with 3'-poly(A) tail, E. coli Poly(A) Polymerase can be used for tailing. The capped and tailed RNA needs to be purified before the RNA transfection experiment.RNase Inhibitor: When configuring the reaction system, you can add 0.5 µl of RNase inhibitor and remove an equal volume of RNase-free water.RNA 5'end labeling: When used for RNA 5'end labeling, the GTP stock solution should be diluted to 1-3 times the mRNA concentration.